There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances. Of particular value are methods for measuring small quantities of pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, antibodies, enzymes and nucleic acids.
The presence of a particular analyte can often be determined by binding methods which exploit the high degree of specificity which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which has been attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting a material of interest. A preferred label should be inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without changing the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations. Radioactive labels are, however, expensive, hazardous, and their use requires sophisticated equipment and trained personnel.
Another means of label detection is electrochemiluminescence, voltage is applied to an electrode, triggering a cyclical oxidation-reduction reaction of a metallo-complex resulting in the generation of light. Because no exciting light is used and only the excited state of the metal ion-ligand complex is produced, the background luminescence is very low.
Electrochemical detection of labels has also generated considerable interest due to its high sensitivity. For example, DNA hybridization has been successfully detected electrochemically using an electrochemical mediator to extract electrons from guanine residues in DNA or RNA and carry them to the electrode, where the mediator is regenerated and can participate in additional electron-transfer events (Ontko et al., Inorg. Chem. 38: 1842-1846 (1999)).
Labels that are detectable using fluorescence spectroscopy are of particular interest, because they are safer than radioactive labels and there is a large number of such labels that are known in the art. However, many fluorescent labels suffer from a number of shortcomings, including a small Stokes shift (absorption and emission curves overlap), a lack of photostability, a decrease in emission due to self-quenching, and the requirement of long excitation wavelengths to get near-IR emission. Thus, there is wide interest in photostable fluorescent labels with a large Stokes shift, that are not self quenching, have long fluorescent lifetimes, and have useful redox properties.
Considerable interest has been sparked by luminescent metal ion complex labels that are photostable, have a large Stokes shift, are not self quenching, have long fluorescent times, and have useful redox properties. Luminescent metal ion complex probes have employed metal ions such as ruthenium (Ru), osmonium (Os) and rhenium (Re). Ruthenium has been the most widely studied metal ion for use in luminescent metal ion complex probes. Ruthenium is a transition metal typically in the +2 oxidation state when forming transition metal complexes. Ru-complexes are octahedral with 6 coordination sites. Ruthenium trisbipyridyl (Ru(bpy)3) has been widely studied by photochemists the last 40 years for such applications as solar cells and artificial photosynthesis.
The absorption spectra of ruthenium complexes are a result of several types of transitions: metal centered, ligand centered, and metal-to-ligand charge transfer at around 450 nm. Emission of Ru(bpy)3 is at ca. 650 nm and the lifetime is ca. 1 μs. Ruthenium complexes are normally very stable and the ligand-metal bonds are covalent in character. As a result, the absorption and emission spectra are very sensitive to changes in the ligands. Moreover, the redox chemistry of ruthenium complexes is well known and often reversible between Ru+1, Ru+2 and Ru+3 states.
There is a growing demand for biological and chemical probes with ruthenium complex labels. Due to the many useful properties of Ru-complexes (long lifetime, Stokes-shifted emission, electrochemistry, intercalation), several types of assays are possible with various detection strategies. For example, Ru-complexes may be used in anisotropy, electrochemiluminescence (e.g. the ORIGEN system from IGEN, Inc.), molecular light switching (see, e.g., Ossipov et al., JACS, 123: 3551-3562 (2001)), electrochemical inter-molecular studies (Tierney et al., J. Phys. Chem. B, 104: 7574-7576 (2001)), electrochemical detection (H. H. Thorp, Trends in Biotechnology 16: 117-121 (1998)) and well known luminescence detection methods such as time-resolved fluorescence and fluorescence polarization.
Accordingly, strategies have been developed and are currently being sought to label biomolecules (e.g., oligonucleotides and peptides) with Ru-complexes. For example, Ru chelate active esters are commercially available and have been used to post synthetically label oligonucleotides. However, this labeling requires a 5-10 fold stoichiometric excess of the Ru(bpy)3-active ester labeling reagent and has many inherent disadvantages, including laborious separation of the labeling reagent from both the labeled and unlabeled oligonucleotides.
Another method involves the use of a nucleoside phosphoramidite comprising a Ru-complex (Khan et al., Inorg. Chem., 38: 3922-3925 (1999)). The method incorporates the phosphoramidite comprising a Ru-complex into an oligonucleotide using solid phase synthesis protocols. However, these methods do not allow the metal complex label to be incorporated on the 3′ end of the oligonucleotide. In addition, the efficiency for coupling the phosphoramidite Ru-complex to the oligonucleotide is poor.
In yet another method, the Ru-complex is added to a nucleic acid after deprotection of the base and cleavage from the solid support. (Tor et al., JACS, 124: 3749-3762 (2002)). However, this method exposes ruthenium coordination sites, such as the N7 guanine, which may result in non-specific binding of the ruthenium.
Thus, there is a need in the art for a more efficient method of making luminescent metal ion complexes. The present invention fills these and other needs in the art.